万寿菊番茄红素β-环化酶基因及其启动子克隆和功能分析

发布时间：2018-06-13 16:46

本文选题：万寿菊 + 番茄红素β-环化酶基因　；参考：《中国农业科学》2017年24期

【摘要】：【目的】克隆万寿菊番茄红素β-环化酶基因和其启动子,进行生物学信息分析,并预测启动子功能,为万寿菊类胡萝卜素的代谢机制和叶黄素含量的调控提供参考依据。【方法】通过RT-PCR技术从万寿菊总RNA中克隆得到TeLCYb的cDNA序列;应用生物学方法分析其DNA序列及其编码蛋白质序列特征,使用DNAMAN和在线软件Box Shade对其氨基酸序列同源性比对分析,并利用MEGA6.0构建进化树,分析其亲缘关系;根据其cDNA序列利用FPNI-PCR法克隆其启动子序列,利用Plant Care在线数据库分析万寿菊TeLCYb启动子调控元件,构建启动子缺失表达载体pTeLCYb(-1969)∷GUS和pTeLCYb(-1140)∷GUS,利用农杆菌介导法侵染烟草,以GUS为报告基因研究不同调控元件的活性。【结果】从万寿菊中成功克隆TeLCYb,生物信息学分析发现,其全长共1 865 bp,开放阅读框为1 527 bp,编码508个氨基酸,氨基酸序列同源比对结果表明其与西洋蒲公英和菊花的同源性最高,且具有LCYb蛋白质特有的保守功能位点和特征多肽序列,在进化上与菊科单独聚为一枝。在已知基因序列的基础上获得长度为1 806 bp的TeLCYb启动子序列,生物学信息分析表明:该启动子除包含核心启动子元件TATA-box、CAAT-box外,还含有多种光应答元件和激素响应元件,其中光响应元件有13个,激素响应元件有5个,此外还具有MYBHv1结合位点元件、耐热响应元件、生理调控作用元件等顺式调控元件。GUS化学组织染色结果显示不同长度启动子均能够驱动GUS在茎、叶、花药及柱头中表达,但不同组织GUS染色蓝色斑点深度不同,其中花器官中花药和柱头中GUS酶活性最强;相对于启动子pTeLCYb(-1969),pTeLCYb(-1140)启动子还能够驱动GUS在根、叶和花萼中特异性表达,且各组织中GUS酶活性明显强于启动子pTeLCYb(-1969)的GUS化学组织染色结果。【结论】不同长度TeLCYb启动子均能驱动下游GUS的表达,但启动子的作用部位和作用强度存在差异,推测在ATG上游1 140 bp和164 bp区间的光响应元件可能具有增强子的功能,而全长启动子特有的激素响应元件和热响应元件可能具有抑制或降低启动子功能的作用。
[Abstract]:[objective] to clone lycopene 尾 -cyclase gene and its promoter of Tagetes marigold, analyze the biological information and predict the function of the promoter. [methods] the cDNA sequence of TeLCYb was cloned from total RNA of marigold by RT-PCR. The DNA sequence and its coding protein sequence were analyzed by biological method. The amino acid sequence homology was analyzed by DNAMAN and box Shade, and the phylogenetic relationship was analyzed by using MEGA 6.0 to construct an evolutionary tree. According to its cDNA sequence, the promoter sequence was cloned by FPNI-PCR, and the promoter regulator element of TeLCYb promoter was analyzed by Plant Care online database. The promoter-deletion expression vectors pTeLCYb-1969) -GUS and pTeLCYb-1140) -GUSwere constructed, and Agrobacterium tumefaciens were used to infect tobacco. The activity of different regulatory elements was studied by using Gus as a reporter gene. [results] TeLCYb was cloned successfully from marigold. Bioinformatics analysis showed that the total length of TeLCYb was 1 865 BP, and the open reading frame was 1 527 BP, encoding 508 amino acids. The results of amino acid sequence homology alignment showed that it had the highest homology with dandelion and chrysanthemum, and had the unique conserved functional site and characteristic polypeptide sequence of LCYb protein. A 1 806 BP TeLCYb promoter sequence was obtained on the basis of the known gene sequence. The biological information analysis showed that the promoter contained not only the core promoter TATA-box CAAT-box, but also a variety of light responders and hormone response elements. Among them, there are 13 light response elements, 5 hormone response elements, and MYBHv1 binding site element, heat resistant response element. The expression of Gus in stem, leaf, anther and stigma were all driven by different length promoters, but the depth of blue spots in different tissues was different. The activity of Gus in anther and stigma was the highest in floral organs, and the specific expression of Gus in root, leaf and calyx was also driven by the promoter pTeLCYb-1969pTeLCYb-1140). The activity of Gus was stronger than that of pTeLCYb-1969. [conclusion] different lengths of TeLCYb promoter can drive the expression of Gus downstream, but the site and intensity of the promoter are different. It is inferred that the photoresponse elements in the upper reaches of ATG (1 140 BP and 164 BP) may have the function of enhancer, while the hormone response element and the thermal response element which are specific to the full-length promoter may inhibit or decrease the function of the promoter.
【作者单位】：华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室/农业部华中都市农业重点实验室(试运行);
【基金】：国家自然科学基金(31672181)
【分类号】：S681.9

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